A Method for Assessing Transduction Efficiency and/or Specificity of Vectors at Single Cell Level

Abstract

Disclosed is a method for assessing the transduction efficiency and/or specificity of vectors at single cell level.

1 . A method for assessing the transduction efficiency and/or specificity of vectors at single cell level, said method comprising:
 a) providing a plurality of different vectors,   b) transducing a heterogeneous population of cells with the plurality of different vectors;   c) partitioning the heterogeneous population of cells into a plurality of compartments, wherein each compartment comprises a single cell from the heterogeneous population of cells;   d) subjecting each partitioned cell to nucleotide sequencing; and   e) detecting the presence of the any one or more of the different vectors in each partitioned cell.   
     
     
         2 . The method of  claim 1 , wherein the method further comprises:
 (f) classifying each partitioned cell into a specific cell type based on gene expression patterns and/or epigenetic features of said cell, as determined using sequencing results obtained in step d).   
     
     
         3 . The method of  claim 1 , wherein the transduction efficiency of a specific vector against a specific cell type is determined by a percentage of cells of the specific cell type which have been detected positive for the presence of the specific vector; and/or wherein the transduction efficiency of a specific vector against a specific cell type is assessed by comparing frequencies with which the presence of said specific vector is detected in the cells of said specific cell type, against frequencies with which the presence of another vector is detected in the cells of said specific cell type; and/or wherein the transduction efficiency of a specific vector against a specific cell type is assessed by comparing the frequencies with which the presence of said specific vector is detected in the cells of said specific cell type, against the frequencies with which the presence of another vector is detected in the cells of said specific cell type. 
     
     
         4 .- 5 . (canceled) 
     
     
         6 . The method of  claim 1 , wherein each of the plurality of different vectors comprises an oligonucleotide barcode sequence, wherein the barcode sequence is different between any two different vectors; and/or wherein the barcode sequence is located on an expression cassette in the vector, wherein the expression of the cassette results in the production of an RNA molecule comprising the barcode sequence, wherein the RNA molecule further comprises a polyadenylation tail. 
     
     
         7 . (canceled) 
     
     
         8 . The method of  claim 6 , wherein the barcode sequence is located on a region of the RNA molecule which allows the barcode sequence to be sequenced. 
     
     
         9 . The method of  claim 8 , wherein the barcode sequence is within a distance of 98 nucleotides from the polyadenylation tail. 
     
     
         10 . The method of  claim 6 , wherein the barcode sequence is 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 nucleotides in length; and/or wherein the barcode sequence is 8 nucleotides in length. 
     
     
         11 . (canceled) 
     
     
         12 . The method of  claim 1 , wherein each of the plurality of different vectors comprises a marker polynucleotide, wherein the marker polynucleotide is different between any two different vectors; and wherein the marker polynucleotide encodes for one or more proteins, said one or more proteins when expressed form a protein envelope which encapsulate the marker polynucleotide, so that after transfection of the vector, each marker polynucleotide is encapsulated by the one or more proteins which the marker polynucleotide encodes for; and/or wherein the marker polynucleotide is located on an expression cassette in the vector, wherein the expression of the cassette results in the production of an RNA molecule comprising the marker polynucleotide, wherein the RNA molecule further comprises a polyadenylation tail; and/or wherein the marker polynucleotide is a viral-capsid-encoding gene, wherein the capsid expressed by the marker polynucleotide encapsulates the marker polynucleotide. 
     
     
         13 .- 14 . (canceled) 
     
     
         15 . The method of  claim 12 , wherein the viral-capsid-encoding gene is specifically an AAV-capsid-encoding gene. 
     
     
         16 . The method of  claim 1 , wherein step e) comprises detecting the presence of one or more marker sequences specific to each different vector; wherein when each vector comprises a unique barcode sequence, said one or more marker sequences comprise the barcode sequence; wherein when each vector comprises a unique marker polynucleotide, said one or more marker sequences comprise the marker polynucleotide. 
     
     
         17 . The method of  claim 16 , wherein step e) comprises matching the sequence reads obtained in step d) with a reference data set. 
     
     
         18 . The method of  claim 17 , wherein the reference data set comprises the genomes and/or the transcriptomes of the plurality of different viral vectors, and/or the barcodes comprised in the plurality of different viral vectors, and/or the marker polynucleotides comprised in the plurality of different viral vectors. 
     
     
         19 . The method of  claim 1 , wherein the compartments are oil droplets. 
     
     
         20 . The method of  claim 1 , wherein the nucleotide sequencing is RNA sequencing and/or DNA sequencing. 
     
     
         21 . (canceled) 
     
     
         22 . The method of  claim 1 , wherein the vectors are selected from the group consisting of: a viral vector, a pseudo-virus vector, a virus-like particle vector, a liposome vector, an exosome vector, a nanoparticle, and combinations thereof; wherein the vectors comprise DNA, RNA, modified RNA, modified DNA, or combinations thereof; and/or wherein the vectors comprise viral vectors, wherein the viral vectors are selected from the group consisting of: an adenoviral vector, an Adeno-associated virus (AAV) vector, a lentiviral vector, a coronavirus vector, an enterovirus vector, a retroviral vector, or a combination thereof. 
     
     
         23 . (canceled) 
     
     
         24 . The method of  claim 22 , wherein the viral vectors are AAV vectors and/or wherein the viral vectors are selected from the group consisting of: AAV type 1 (AAV-1), AAV type 2 (AAV-2), AAV type 3 (AAV-3), AAV type 4 (AAV-4), AAV type 5 (AAV-5), AAV type 6 (AAV-6), AAV type 7 (AAV-7), AAV type 8 (AAV-8), AAV type 9 (AAV9), AAV type 10 (AAV10), AAV type 11 (AAV11), AAV type 12 (AAV12), AAV type 13 (AAV13), rh10, AAVDJ, AAVAnc80, AAV-PHP.S, AAV-PHP.eB, AAV-LK03, AAV2-7m8, AAV variants thereof, and combinations thereof. 
     
     
         25 . (canceled) 
     
     
         26 . The method of  claim 22 , wherein the plurality of different viral vectors comprises viral vectors of different families, viral vectors of different genera, viral vectors of different species, viral vectors of different serotypes, viral vectors thereof carrying different mutations, or combinations thereof. 
     
     
         27 . The method of  claim 1 , wherein the heterogeneous population of cells comprise plant cells, animal cells, fungal cells, or combinations thereof. 
     
     
         28 . The method of  claim 27 , wherein the heterogeneous population of cells comprise mammalian cells; and/or the heterogeneous population of cells comprise human cells; and/or the heterogeneous population of cells are comprised in an animal or human subject when being transduced. 
     
     
         29 .- 32 . (canceled) 
     
     
         33 . The method of  claim 27 , wherein the one or more cultured organoids are selected from the group consisting of ocular organoid, cerebral organoid, epithelial organoid, kidney organoid, lung organoid, pancreas organoid, cardiac organoid, and hepatic organoid.